PROJECT SUMMARY The incidence of melanoma, the deadliest of all skin cancers, is rapidly increasing. Recent FDA-approved targeted and immune therapies, the mainstay of treatment for metastatic melanoma, are constrained by restricted efficacy, drug resistance, and severe toxicities. Thus, improved understanding of the mechanisms driving melanoma progression remains an unmet medical need that could provide effective means of preventing tumor recurrence with improved on-target therapeutic strategies. Advances in next-generation sequencing technologies have uncovered widespread spicing aberrancies across the majority of cancers. Although alternative splicing, predominantly regulated by RNA-binding proteins (RBPs), serves to diversify more than 90% of human protein-coding genes, emerging studies describe novel mechanisms by which this post-transcriptional regulation is hijacked to support oncogenic programs. Recent efforts identify tumor-specific deregulation of the spliceosome complex and/or alternative isoforms with oncogenic or tumor suppressive function. In melanoma, differential mRNA processing in oncogenic drivers has been shown to promote metastasis and resistance to BRAF inhibition. Despite compelling evidence that alternative splicing contributes to melanoma pathogenesis, a comprehensive study of the molecular actors mediating this effect has not been performed. To address this, our lab profiled changes in gene expression and histone marks in primary and metastatic melanoma patient samples, as well as control melanocytes. Of 489 canonical RBPs, 17 RBPs in primary melanoma and 104 RBPs in metastatic melanoma were significantly upregulated relative to healthy controls, with 52 RBPs were significantly upregulated in metastatic sites relative to the primary site of disease. To investigate dependencies on RBPs in melanoma pathogenesis, I have performed a CRISPR-Cas9 negative selection screening approach in human melanoma cell lines. As RBPs exert highly cell- and tissue- specific function through RNA-binding domains (RBDs), single-guide(sg)RNAs were designed against the RBDs of all 489 canonical RBPs. Using this negative-selection approach, I identified hnRNP A1 as an RBP essential to melanoma in vitro. HnRNP A1, a ubiquitously expressed protein and member of the diverse family of heterogeneous ribonucleoproteins, is broadly implicated in RNA processing events, such as splicing, stability, nuclear export, and translation. HnRNP A1 expression was upregulated in melanoma relative to melanocytes, and genetic ablation of hnRNP A1 revealed it to be critical to melanoma cell survival. The proposed work will characterize the requirement for hnRNP A1 in melanoma. This work will elucidate the complex post-transcriptional regulation underlying melanoma pathogenesis and provide a strong rationale for developing novel therapeutic strategies, leveraging this information to improve patient outcomes.